Seasonality provokes a shift of thermal preferences in a temperate lizard,but altitude does not

1. We compared the mean, limits and breadth of the preferred thermal range (PTR) of two Iberian populations of the lizard Psammodromus algirus separated by 700 m altitude in May and July.     

Seed germination ecology of the threatened endemic Iberian <Emphasis Type="Italic">Delphinium fissum</Emphasis> subsp. <Emphasis Type="Italic">sordidum</Emphasis> (Ranunculaceae)

Seeds of Delphinium fissum subsp. sordidum are physiologically dormant at maturity, with underdeveloped embryos; thus they have morphophysiological dormancy (MPD). The aims of this study were to determine the requirements for embryo growth, dormancy break and germination, to characterise the type of seed dormancy and to evaluate the effects of light, seed age, pollination mechanism, and inter-annual and inter-population variability on germinative ability. After 3 months of incubation at 5°C (cold stratification) in darkness conditions, the mean embryo length increased from 5.6 to 2.07 mm, with 76% of seeds germinating. Conversely, embryos of seeds incubated during 3 months at 20/7 or 28/14°C hardly grew and no germination was recorded. Since cold stratification was the only requirement for the loss of MPD, and both dry storage in laboratory conditions and warm stratification prior to cold stratification shortened the cold stratification period required for germination, it could be concluded that D. fissum subsp. sordidum seeds have intermediate complex MPD. Cold stratification and incubation in darkness conditions promoted higher germination percentages than those in light. In addition, germinative ability increased with seed age up to 8 months (reaching 96% at 5°C in darkness), showed a pronounced inter-annual and inter-population variability, as well as a significant decrease in seeds coming from pollination by geitonogamy. High temperatures (25/10 or 28/14°C) induced seeds to secondary dormancy, so seedling emergence in the greenhouse was restricted to February–March. The requirements for dormancy break and germination reflect an adaptation to trigger germination in late winter. This study is the first one to document a gradual increase in germination percentage with seed age for plant species with intermediate complex MPD.     

Chinese hamster apurinic/apyrimidinic endonuclease (chAPE1) expressed in sf9 cells reveals that its endonuclease activity is regulated by phosphorylation

Apurinic/apyrimidinic endonuclease (APE), an essential DNA repair enzyme, initiates the base excision repair pathway by creating a nick 5' to an abasic site in double-stranded DNA. Although the Chinese hamster ovary cells remain an important model for DNA repair studies, the Chinese hamster APE (chAPE1) has not been studied in vitro in respect to its kinetic characteristics. Here we report the results of a kinetic study performed on cloned and overexpressed enzyme in sf9 cells. The kinetic parameters were fully compatible with the broad range of kinetic parameters reported for the human enzyme. However, the activity measures depended on the time point of the culture. We applied inductivity coupled plasma spectrometry to measure the phosphorylation level of chAPE1. Our data showed that a higher phosphorylation of chAPE1 in the expression host was correlated to a lower endonuclease activity. The phosphorylation of a higher activity batch of chAPE1 by casein kinase II decreased the endonuclease activity, and the dephosphorylation of chAPE1 by lambda phosphatase increased the endonuclease activity. The exonuclease activity of chAPE1 was not observed in our kinetic analysis. The results suggest that noticeable divergence in reported activity levels for the human APE1 endonuclease might be caused by unaccounted phosphorylation. Our data also demonstrate that only selected kinases and phosphatases exert regulatory effects on chAPE1 endonuclease activity, suggesting further that this regulatory mechanism may function in vivo to turn on and off the function of this important enzyme in different organisms.     

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition ^1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells ^1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components ³. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration ⁴. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands ^5-8. However, not until recently have any chemoattractants of trunk NCCs been identified ⁹. Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere¹⁰). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.     

Conformational changes of yeast plasma membrane H(+)-ATPase during activation by glucose: role of threonine-912 in the carboxy-terminal tail

Yeast Pma1 H(+)-ATPase, which belongs to the P-type family of cation-transporting ATPases, is activated up to 10-fold by growth on glucose, and indirect evidence has linked the activation to Ser/Thr phosphorylation within the C-terminal tail. We have now used limited trypsinolysis to map glucose-induced conformational changes throughout the 100 kDa ATPase. In the wild-type enzyme, trypsin cleaves first at Lys-28 and Arg-73 in the extended N-terminal segment (sites T1 and T2); subsequent cleavages occur at Arg-271 between the A domain and M3 (site T3) and at Lys-749 or Lys-754 in the M6-M7 cytoplasmic loop (site T4). Activation by glucose leads to a striking increase in trypsin sensitivity. At the C-terminal end of the protein, the Arg- and Lys-rich tail is shielded from trypsin in membranes from glucose-starved cells (GS) but becomes accessible in membranes from glucose-metabolizing cells (GM). In the presence of orthovanadate, Lys-174 at the boundary between M2 and the A domain also becomes open to cleavage in GM but not GS samples (site T5). Significantly, this global conformational change can be suppressed by mutations at Thr-912, a consensus phosphorylation site near the C-terminus. Substitution by Ala at position 912 leads to a GS-like (trypsin-resistant) state, while substitution by Asp leads to a GM-like (trypsin-sensitive) state. Thus, the present results help to dissect the intramolecular movements that result in glucose activation.     

Molecular phylogeny of the Acre clade (Crassulaceae): Dealing with the lack of definitions for Echeveria and Sedum

The phylogenetic relationships within many clades of the Crassulaceae are still uncertain, therefore in this study attention was focused on the “Acre clade”, a group comprised of approximately 526 species in eight genera that include many Asian and Mediterranean species of Sedum and the majority of the American genera (Echeveria, Graptopetalum, Lenophyllum, Pachyphytum, Villadia, and Thompsonella). Parsimony and Bayesian analyses were conducted with 133 species based on nuclear (ETS, ITS) and chloroplast DNA regions (rpS16, matK). Our analyses retrieved four major clades within the Acre clade. Two of these were in a grade and corresponded to Asian species of Sedum, the rest corresponded to a European–Macaronesian group and to an American group. The American group included all taxa that were formerly placed in the Echeverioideae and the majority of the American Sedoideae. Our analyses support the monophyly of three genera – Lenophyllum, Thompsonella, and Pachyphytum; however, the relationships among Echeveria, Sedum and the various segregates of Sedum are largely unresolved. Our analyses represents the first broad phylogenetic framework for Acre clade, but further studies are necessary on the groups poorly represented here, such as the European and Asian species of Sedum and the Central and South American species of Echeveria.     

Mitogen-activated protein kinases modulate H(2)O(2)-induced apoptosis in primary rat alveolar epithelial cells

Increasing evidence suggests a role for apoptosis in the maintenance of the alveolar epithelium under normal and pathological conditions. However, the signaling pathways modulating alveolar type II (ATII) cell apoptosis remain poorly defined. Here we investigated the role of MAPKs as modulators of oxidant-mediated ATII cell apoptosis using in vitro models of H(2)O(2)-stress. H(2)O(2), delivered either as a bolus or as a flux, lead to time- and concentration-dependent increases in ATII cells apoptosis. Increased apoptosis in primary rat ATII cells was detected at H(2)O(2) concentrations and production rates in the physiological range (1 microM) and peaked at 100 microM H(2)O(2). Immortalized rat lung epithelial cells (RLE), in contrast, required millimolar concentration of H(2)O(2) for maximal responses. H(2)O(2)-induced apoptosis was preceded by rapid activation of all three classes of mitogen-activated protein kinases (MAPKs): ERK, JNK, and p38. Specific inhibition of JNK using antisense oligonucleotides and ERK and p38 using PD98059 or SB202190, respectively, indicated a pro-apoptotic role for JNK pathway and an anti-apoptotic role for ERK- and p38-initiated signaling events. Our data show that the balance between the activation of JNK, ERK, and p38 is a critical determinant of cell fate, suggesting that pharmacological interventions on the MAPK pathways may be useful in the treatment of oxidant-related lung injury.     

Hypertriglyceridemia Influences the Degree of Postprandial Lipemic Response in Patients with Metabolic Syndrome and Coronary Artery Disease: From the Cordioprev Study

Objective

To determine whether metabolic syndrome traits influence the postprandial lipemia response of coronary patients, and whether this influence depends on the number of MetS criteria.

Materials and Methods

1002 coronary artery disease patients from the CORDIOPREV study were submitted to an oral fat load test meal with 0.7 g fat/kg body weight (12% saturated fatty acids, 10% polyunsaturated fatty acids, 43% monounsaturated fatty acids), 10% protein and 25% carbohydrates. Serial blood test analyzing lipid fractions were drawn at 0, 1, 2, 3 and 4 hours during the postprandial state. Total and incremental area under the curves of the different postprandial parameters were calculated following the trapezoid rule to assess the magnitude of change during the postprandial state

Results

Postprandial lipemia response was directly related to the presence of metabolic syndrome. We found a positive association between the number of metabolic syndrome criteria and the response of postprandial plasma triglycerides (p<0.001), area under the curve of triglycerides (p<0.001) and incremental area under the curve of triglycerides (p<0.001). However, the influence of them on postprandial triglycerides remained statistically significant only in those patients without basal hypertriglyceridemia. Interestingly, in stepwise multiple linear regression analysis with the AUC of triglycerides as the dependent variable, only fasting triglycerides, fasting glucose and waist circumference appeared as significant independent (P<0.05) contributors. The multiple lineal regression (R) was 0.77, and fasting triglycerides showed the greatest effect on AUC of triglycerides with a standardized coefficient of 0.75.

Conclusions

Fasting triglycerides are the major contributors to the postprandial triglycerides levels. MetS influences the postprandial response of lipids in patients with coronary heart disease, particularly in non-hypertriglyceridemic patients.     

Thermal limits for the establishment and growth of populations of the invasive apple snail <Emphasis Type="Italic">Pomacea canaliculata</Emphasis>

Pomacea canaliculata is a South American freshwater snail considered as one of the world’s worst invasive alien species. A temperature of around 25 °C has usually been considered to be optimal for rearing P. canaliculata. Nevertheless, snails have not been reared under a wide range of temperatures to reveal the optimum for performance in terms of population increase. We investigated the effect of temperature on growth, survival and reproduction, estimating demographic parameters for P. canaliculata in the wide range of temperatures at which these snails are active (15–35 °C). No reproductive activity was evidenced for the snails reared at 15 °C, probably explained by the small sizes attained at this temperature. Temperatures above 25 °C did not promote a significant acceleration in growth so higher temperatures will not result in a reduction in time to reach maturity. In fact, snails from 25 and 30 °C began reproduction at the same age. We report here for the first time a detrimental effect of high temperatures that provoked a significant decrease in the contribution of snails to the next generation: the viability of eggs from the snails reared at 30 °C was very low and the snails exposed to a constant water temperature of 35 °C were unable to produce eggs. Our findings reveal a new environmental constraint that could be a determinant of the range limits of this species in invaded regions, especially during the coming decades, anticipating the scenario predicted from global warming.